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Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

Bottom Line: We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins.Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands.Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

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Related in: MedlinePlus

Interaction of GIV with EGFR determines the profile of other SH2 adaptors recruited to the ligand-activated receptor. (a) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1173 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with q constant amount (6 μg) of His-GIV-CT and increasing amounts of His-SHP-1 as indicated. Bound proteins were analyzed for His-SHP1 and His-GIV-CT by IB with His. (b) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1148 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with a constant amount (2 μg) of His-Shc1 and increasing amounts of His-GIV-CT as indicated. Bound proteins were analyzed for His-Shc1 and His-GIV-CT by IB with His. (c) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. EGFR was immunoprecipitated from these lysates as in Figure 5d. Immunoprecipitates (left) and lysates (right) were analyzed for receptor phosphorylation and various SH2 adaptors by IB. Compared to HeLa GIV-WT cells, in HeLa GIV-RL cells, recruitment of SHP1 and Shc1 to ligand-activated EGFR is enhanced, autophosphorylation of EGFR is reduced, and recruitment of p85α (PI3K) and Grb2 is suppressed. (d) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. Equal aliquots of whole-cell lysates were analyzed for phospho-Akt (pAkt), ERK (pERK1/2), and tubulin by IB. (e) Working model. A schematic summarizing the sequence of events triggered by the binding of GIV's SH2-like domain to EGFR. Left, in the absence of GIV, upon ligand stimulation, EGFR autophosphorylation is triggered at Y1148 and Y1173, which serve as sites of adaptor recruitment for Shc and SHP-1, respectively. Shc triggers activation of the MAPK/ERK pathway, and activated SHP-1 dephosphorylates EGFR and down-regulates receptor signaling. Right, in the presence of GIV, EGFR autophosphorylation sites pY1148 and pY1173 are recognized and approached by a partially structured SH2-like domain in GIV's C-terminus. Close proximity to EGFR facilitates efficient phosphorylation of GIV on critical tyrosines within a partially structured SH2 domain before/simultaneously with folding into a SH2-like module that stably docks onto autophosphorylated EGFR tail. Such docking competes with Shc for Y1148 and with SHP-1 for Y1173, thereby displacing and antagonizing signaling via both adaptors. Once GIV-SH2 is recruited to activated EGFR, GIV triggers two parallel pathways that were previously shown to synergistically activate Akt: a) GIV's phosphotyrosines bind p85α (class Ia PI3K) and activate PI3K/Akt signaling (Lin et al., 2011), and b) GIV's GEF motif activates Gαi in close proximity to activated EGFR and releases “free” Gβϒ, which directly binds p110 (class 1b PI3K) and activates PI3K/Akt signaling (Garcia-Marcos et al., 2009).
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Figure 6: Interaction of GIV with EGFR determines the profile of other SH2 adaptors recruited to the ligand-activated receptor. (a) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1173 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with q constant amount (6 μg) of His-GIV-CT and increasing amounts of His-SHP-1 as indicated. Bound proteins were analyzed for His-SHP1 and His-GIV-CT by IB with His. (b) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1148 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with a constant amount (2 μg) of His-Shc1 and increasing amounts of His-GIV-CT as indicated. Bound proteins were analyzed for His-Shc1 and His-GIV-CT by IB with His. (c) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. EGFR was immunoprecipitated from these lysates as in Figure 5d. Immunoprecipitates (left) and lysates (right) were analyzed for receptor phosphorylation and various SH2 adaptors by IB. Compared to HeLa GIV-WT cells, in HeLa GIV-RL cells, recruitment of SHP1 and Shc1 to ligand-activated EGFR is enhanced, autophosphorylation of EGFR is reduced, and recruitment of p85α (PI3K) and Grb2 is suppressed. (d) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. Equal aliquots of whole-cell lysates were analyzed for phospho-Akt (pAkt), ERK (pERK1/2), and tubulin by IB. (e) Working model. A schematic summarizing the sequence of events triggered by the binding of GIV's SH2-like domain to EGFR. Left, in the absence of GIV, upon ligand stimulation, EGFR autophosphorylation is triggered at Y1148 and Y1173, which serve as sites of adaptor recruitment for Shc and SHP-1, respectively. Shc triggers activation of the MAPK/ERK pathway, and activated SHP-1 dephosphorylates EGFR and down-regulates receptor signaling. Right, in the presence of GIV, EGFR autophosphorylation sites pY1148 and pY1173 are recognized and approached by a partially structured SH2-like domain in GIV's C-terminus. Close proximity to EGFR facilitates efficient phosphorylation of GIV on critical tyrosines within a partially structured SH2 domain before/simultaneously with folding into a SH2-like module that stably docks onto autophosphorylated EGFR tail. Such docking competes with Shc for Y1148 and with SHP-1 for Y1173, thereby displacing and antagonizing signaling via both adaptors. Once GIV-SH2 is recruited to activated EGFR, GIV triggers two parallel pathways that were previously shown to synergistically activate Akt: a) GIV's phosphotyrosines bind p85α (class Ia PI3K) and activate PI3K/Akt signaling (Lin et al., 2011), and b) GIV's GEF motif activates Gαi in close proximity to activated EGFR and releases “free” Gβϒ, which directly binds p110 (class 1b PI3K) and activates PI3K/Akt signaling (Garcia-Marcos et al., 2009).

Mentions: We previously demonstrated (Ghosh et al., 2010) that GIV enhances receptor autophosphorylation, affects the recruitment of key signaling adaptors to the receptor tail, and alters several major EGF signaling pathways such that they are either selectively amplified (e.g., PI3K/Akt) or attenuated (e.g., mitogen-activated protein kinase/extracellular signal-regulated kinase [MAPK/ERK]) via poorly understood mechanisms. We reasoned that by virtue of its ability to directly bind pY1148 and 1173 on EGFR tail, the newly identified SH2-like domain in GIV may shape EGF signaling by affecting the profile of SH2 adaptor recruitment to the ligand-activated receptor tail. We focused on Shc1 and SHP1, two SH2 adaptors that are major players in shaping EGF signaling (Keilhack et al., 1998; Sakaguchi et al., 1998), because they bind EGFR predominantly at the sequences flanking pY1148 and pY1173: Shc1 binds primarily to pY1148 and weakly to pY1173 (Okabayashi et al., 1994), whereas SHP1 binds primarily to pY1173 and weakly to pY1148 (Keilhack et al., 1998). In GST pull-down assays with immobilized phosphorylated GST-EGFR tail pY1173 peptide, increasing amounts of His-SHP-1, and constant amounts of His-tagged GIV-CT proteins in solution, we found that increased binding of SHP1 coincided with decreased binding of GIV-CT to EGFR phosphopeptide (Figure 6a), indicating that SHP1 and GIV-CT compete for pY1173 on EGFR tail. Similarly, in GST pull-down assays with immobilized phosphorylated GST-EGFR tail pY1148 peptide, increasing amounts of His-tagged GIV-CT, and constant amounts of Shc1 proteins in solution, we found that increased binding of GIV coincided with decreased binding of Shc1 to EGFR phosphopeptide (Figure 6b), indicating that Shc1 and GIV-CT compete for pY1148 on EGFR tail. To determine whether such competition occurs in cells, we evaluated the profile of SH2-adaptor recruitment to the EGFR tail in GIV-WT versus GIV-RL HeLa cells by immunoprecipitating the receptor and analyzing the receptor-bound complexes by immunoblotting (IB). We found that the profile of adaptor recruitment was significantly different in the two cell lines. GIV was recruited to ligand-activated EGFR in GIV-WT but not in GIV-RL cells (Figure 6c). When GIV failed to be recruited to EGFR in GIV-RL cells, recruitment of p85α (PI3K) and Grb2 was also suppressed but recruitment of SHP1 and Shc1 was enhanced. Consistent with the enhanced recruitment of SHP1, in GIV-RL cells, we observe that activation of SHP1, as determined by the abundance of pY536SHP1 (Figure 6c, Lysates), and dephosphorylation of key autophosphorylation sites in EGFR (Figure 6c, Immunoprecipitates) were enhanced. Together these results demonstrate that binding of GIV-SH2 competes with SHP1 and Shc1 for binding to two major sites of autophosphorylation on EGFR.


Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

Interaction of GIV with EGFR determines the profile of other SH2 adaptors recruited to the ligand-activated receptor. (a) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1173 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with q constant amount (6 μg) of His-GIV-CT and increasing amounts of His-SHP-1 as indicated. Bound proteins were analyzed for His-SHP1 and His-GIV-CT by IB with His. (b) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1148 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with a constant amount (2 μg) of His-Shc1 and increasing amounts of His-GIV-CT as indicated. Bound proteins were analyzed for His-Shc1 and His-GIV-CT by IB with His. (c) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. EGFR was immunoprecipitated from these lysates as in Figure 5d. Immunoprecipitates (left) and lysates (right) were analyzed for receptor phosphorylation and various SH2 adaptors by IB. Compared to HeLa GIV-WT cells, in HeLa GIV-RL cells, recruitment of SHP1 and Shc1 to ligand-activated EGFR is enhanced, autophosphorylation of EGFR is reduced, and recruitment of p85α (PI3K) and Grb2 is suppressed. (d) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. Equal aliquots of whole-cell lysates were analyzed for phospho-Akt (pAkt), ERK (pERK1/2), and tubulin by IB. (e) Working model. A schematic summarizing the sequence of events triggered by the binding of GIV's SH2-like domain to EGFR. Left, in the absence of GIV, upon ligand stimulation, EGFR autophosphorylation is triggered at Y1148 and Y1173, which serve as sites of adaptor recruitment for Shc and SHP-1, respectively. Shc triggers activation of the MAPK/ERK pathway, and activated SHP-1 dephosphorylates EGFR and down-regulates receptor signaling. Right, in the presence of GIV, EGFR autophosphorylation sites pY1148 and pY1173 are recognized and approached by a partially structured SH2-like domain in GIV's C-terminus. Close proximity to EGFR facilitates efficient phosphorylation of GIV on critical tyrosines within a partially structured SH2 domain before/simultaneously with folding into a SH2-like module that stably docks onto autophosphorylated EGFR tail. Such docking competes with Shc for Y1148 and with SHP-1 for Y1173, thereby displacing and antagonizing signaling via both adaptors. Once GIV-SH2 is recruited to activated EGFR, GIV triggers two parallel pathways that were previously shown to synergistically activate Akt: a) GIV's phosphotyrosines bind p85α (class Ia PI3K) and activate PI3K/Akt signaling (Lin et al., 2011), and b) GIV's GEF motif activates Gαi in close proximity to activated EGFR and releases “free” Gβϒ, which directly binds p110 (class 1b PI3K) and activates PI3K/Akt signaling (Garcia-Marcos et al., 2009).
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Figure 6: Interaction of GIV with EGFR determines the profile of other SH2 adaptors recruited to the ligand-activated receptor. (a) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1173 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with q constant amount (6 μg) of His-GIV-CT and increasing amounts of His-SHP-1 as indicated. Bound proteins were analyzed for His-SHP1 and His-GIV-CT by IB with His. (b) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1148 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with a constant amount (2 μg) of His-Shc1 and increasing amounts of His-GIV-CT as indicated. Bound proteins were analyzed for His-Shc1 and His-GIV-CT by IB with His. (c) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. EGFR was immunoprecipitated from these lysates as in Figure 5d. Immunoprecipitates (left) and lysates (right) were analyzed for receptor phosphorylation and various SH2 adaptors by IB. Compared to HeLa GIV-WT cells, in HeLa GIV-RL cells, recruitment of SHP1 and Shc1 to ligand-activated EGFR is enhanced, autophosphorylation of EGFR is reduced, and recruitment of p85α (PI3K) and Grb2 is suppressed. (d) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. Equal aliquots of whole-cell lysates were analyzed for phospho-Akt (pAkt), ERK (pERK1/2), and tubulin by IB. (e) Working model. A schematic summarizing the sequence of events triggered by the binding of GIV's SH2-like domain to EGFR. Left, in the absence of GIV, upon ligand stimulation, EGFR autophosphorylation is triggered at Y1148 and Y1173, which serve as sites of adaptor recruitment for Shc and SHP-1, respectively. Shc triggers activation of the MAPK/ERK pathway, and activated SHP-1 dephosphorylates EGFR and down-regulates receptor signaling. Right, in the presence of GIV, EGFR autophosphorylation sites pY1148 and pY1173 are recognized and approached by a partially structured SH2-like domain in GIV's C-terminus. Close proximity to EGFR facilitates efficient phosphorylation of GIV on critical tyrosines within a partially structured SH2 domain before/simultaneously with folding into a SH2-like module that stably docks onto autophosphorylated EGFR tail. Such docking competes with Shc for Y1148 and with SHP-1 for Y1173, thereby displacing and antagonizing signaling via both adaptors. Once GIV-SH2 is recruited to activated EGFR, GIV triggers two parallel pathways that were previously shown to synergistically activate Akt: a) GIV's phosphotyrosines bind p85α (class Ia PI3K) and activate PI3K/Akt signaling (Lin et al., 2011), and b) GIV's GEF motif activates Gαi in close proximity to activated EGFR and releases “free” Gβϒ, which directly binds p110 (class 1b PI3K) and activates PI3K/Akt signaling (Garcia-Marcos et al., 2009).
Mentions: We previously demonstrated (Ghosh et al., 2010) that GIV enhances receptor autophosphorylation, affects the recruitment of key signaling adaptors to the receptor tail, and alters several major EGF signaling pathways such that they are either selectively amplified (e.g., PI3K/Akt) or attenuated (e.g., mitogen-activated protein kinase/extracellular signal-regulated kinase [MAPK/ERK]) via poorly understood mechanisms. We reasoned that by virtue of its ability to directly bind pY1148 and 1173 on EGFR tail, the newly identified SH2-like domain in GIV may shape EGF signaling by affecting the profile of SH2 adaptor recruitment to the ligand-activated receptor tail. We focused on Shc1 and SHP1, two SH2 adaptors that are major players in shaping EGF signaling (Keilhack et al., 1998; Sakaguchi et al., 1998), because they bind EGFR predominantly at the sequences flanking pY1148 and pY1173: Shc1 binds primarily to pY1148 and weakly to pY1173 (Okabayashi et al., 1994), whereas SHP1 binds primarily to pY1173 and weakly to pY1148 (Keilhack et al., 1998). In GST pull-down assays with immobilized phosphorylated GST-EGFR tail pY1173 peptide, increasing amounts of His-SHP-1, and constant amounts of His-tagged GIV-CT proteins in solution, we found that increased binding of SHP1 coincided with decreased binding of GIV-CT to EGFR phosphopeptide (Figure 6a), indicating that SHP1 and GIV-CT compete for pY1173 on EGFR tail. Similarly, in GST pull-down assays with immobilized phosphorylated GST-EGFR tail pY1148 peptide, increasing amounts of His-tagged GIV-CT, and constant amounts of Shc1 proteins in solution, we found that increased binding of GIV coincided with decreased binding of Shc1 to EGFR phosphopeptide (Figure 6b), indicating that Shc1 and GIV-CT compete for pY1148 on EGFR tail. To determine whether such competition occurs in cells, we evaluated the profile of SH2-adaptor recruitment to the EGFR tail in GIV-WT versus GIV-RL HeLa cells by immunoprecipitating the receptor and analyzing the receptor-bound complexes by immunoblotting (IB). We found that the profile of adaptor recruitment was significantly different in the two cell lines. GIV was recruited to ligand-activated EGFR in GIV-WT but not in GIV-RL cells (Figure 6c). When GIV failed to be recruited to EGFR in GIV-RL cells, recruitment of p85α (PI3K) and Grb2 was also suppressed but recruitment of SHP1 and Shc1 was enhanced. Consistent with the enhanced recruitment of SHP1, in GIV-RL cells, we observe that activation of SHP1, as determined by the abundance of pY536SHP1 (Figure 6c, Lysates), and dephosphorylation of key autophosphorylation sites in EGFR (Figure 6c, Immunoprecipitates) were enhanced. Together these results demonstrate that binding of GIV-SH2 competes with SHP1 and Shc1 for binding to two major sites of autophosphorylation on EGFR.

Bottom Line: We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins.Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands.Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

Show MeSH
Related in: MedlinePlus